Garber R L, Gage L P
Cell. 1979 Nov;18(3):817-28. doi: 10.1016/0092-8674(79)90134-x.
We have analyzed the transcription of a cloned silkworm tRNA2Ala gene in germinal vesicle extracts of X. laevis oocytes. The primary transcript was sequenced; it is 98 nucleotides long, beginning with a 5' triphosphate nucleotide and ending in a 3' oligouridine stretch. After transcription for long periods of time, enzymes in the frog extract also process the tRNA2Ala precursor to remove extra 5' and 3' nucleotides and to add a CCA end. The twenty-two extra nucleotides at the 3' end of this precursor are recovered as an intact fragment, implicating a new site of endoribonuclease cleavage in eucaryotic tRNA processing. This enzyme activity has also been demonstrated by reincubation of isolated pre-tRNA2Ala with a germinal vesicle extract. The products of in vitro cleavage are the same as those seen in the transcription reactions. The tRNA2Ala precursor molecules are made faithfully in the system with as few as 6 bp of Bombyx morti DNA upstream of the transcription initiation site of the tRNA2Ala gene. This result narrows down the minimal amount of DNA adjacent to the 5' end of a eucaryotic tRNA gene needed to support proper initiation by RNA polymerase III.
我们分析了家蚕tRNA2Ala基因在非洲爪蟾卵母细胞生发泡提取物中的转录情况。对初级转录本进行了测序；其长度为98个核苷酸，起始于5'三磷酸核苷酸，终止于3'寡聚尿苷序列。长时间转录后，蛙提取物中的酶也会加工tRNA2Ala前体，去除多余的5'和3'核苷酸，并添加一个CCA末端。该前体3'端的22个额外核苷酸以完整片段的形式回收，这意味着在真核tRNA加工过程中有一个新的核糖核酸内切酶切割位点。通过将分离的前体tRNA2Ala与生发泡提取物再次温育，也证明了这种酶活性。体外切割产物与转录反应中观察到的产物相同。在该系统中，tRNA2Ala前体分子的合成非常准确，在tRNA2Ala基因转录起始位点上游仅需6个碱基对的家蚕DNA。这一结果缩小了真核tRNA基因5'端相邻的、支持RNA聚合酶III正确起始所需的最小DNA量。
